Aniline is currently produced at several million tonnes per year from fossil raw materials, e.g. to produce polyurethanes. An aniline source based on renewable resources, also called “bioaniline”, is strongly desired for the chemical industry in order to become independent from fossil resources. More importantly, there is a strong desire to reduce carbon dioxide (CO2) emissions both for the chemical processes as well as by increasing the use of renewable resources in the raw materials. Bioaniline has a high potential of saving CO2 emissions.
The invention further relates to engineering of microorganisms and production of aromatic compounds therefrom. In particular, the invention relates to the field of producing o-aminobenzoate (oAB) from renewable sources, such as e.g. biomass in a suitable recombinant microbial host. Typically a source containing a significant proportion of fermentable sugars is used. These sugars may include polysaccharides such as disaccharides, e.g. sucrose, or trisaccharides, e.g. kestose, as well as C-6 monosaccharides such as glucose, fructose or mannose and C-5 monosaccharides such as xylose and arabinose. A recombinant microbial strain capable of converting sugar to o-aminobenzoate (2-aminobenzoate, or tho-aminobenzoate, o-aminobenzoate, oAB) would enable the production of o-aminobenzoate from a wide range of renewable resources including sugar beet and sugar cane, starch-containing plants such as corn, wheat and rye, as well as lignocellulose e.g. from straw, wood or bagasse.
Currently, there is no renewable or biologically derived source of o-aminobenzoate or the corresponding acid available commercially and no known example of the large-scale biological production of o-aminobenzoate has been described. o-Aminobenzoate is a natural intermediate of the shikimate acid pathway and a precursor for the biosynthesis of the aromatic amino acid L-tryptophane. The biosynthetic pathway to o-aminobenzoate is relatively well understood in both prokaryotes and eukaryotes. A chemical conversion of o-aminobenzoate to aniline can be achieved. Current production methods of aniline rely on chemical synthesis from petroleum-derived raw-materials. Such petroleum-derived raw materials are not renewable as opposed to raw materials which are renewable, such as the renewable resource “biomass”. Several chemical steps involved in the chemical synthesis result in high production costs of the chemicals. The conventional chemical synthesis of aniline can be associated with hazardous intermediates, solvents, and waste products which can have substantial impacts on the environment. Non-specific side-reactions on the aromatic-ring result in the reduction of the product yield. Petroleum-derived raw materials are influenced by cost fluctuations resulting from the global petroleum price.
WO 2013/103894 A1 discloses a method of producing aromatic amines via biologically-derived p-aminobenzoic acid (4-aminobenzoate). However, this document discloses to produce the p-aminobenzoic acid in either E. coli or in S. cerevisiae and fails to recognize the advantages of Corynebacterium glutamicum as a host. In addition, this document does also not disclose how to successfully combine the fermentation process with the downstream chemical process of converting the biologically-derived p-aminobenzoic acid to aromatic amines, e.g. aniline. Regarding the downstream chemical process technology of how to convert chemically or biologically produced the p-aminobenzoic acid this document merely refers to distillation methods without recognizing the advantageous technical benefits of combining this part with the upstream part of providing the p-aminobenzoic acid in form of a continuous process.
A direct fermentation of sugar to aniline as a one-step conversion was thought to be most cost efficient if based on a biosynthesis pathway including an enzymatic, in vivo, decarboxylation of anthranilate to aniline as the final reaction step. Since an aminobenzoate decarboxylase could not successfully be identified or developed through protein engineering, the decarboxylation reaction of anthranilate to aniline could not be carried out by pure enzymatic means. Since such a one-step process was not technically feasible, process alternatives to perform the final reaction step of decarboxylating anthranilate to aniline as the final reaction step were taken into consideration, e.g. by a chemical step, as opposed to an enzymatic step.